Prolonged ingestion of a high fat diet (HFD) is associated with hyperphagia and obesity. Rats and humans fed HFDs exhibit reduced sensitivity to a number of satiety signals known to act via the vagal afferent pathways. Our preliminary studies indicate that these abnormalities may be prevented by blockade of Toll-like receptor 4 signaling pathway or administration of EP2 receptor inhibitor, suggesting medication by prostaglandin evoked by LPS. Patch clamp studies of nodose ganglia (NG) neurons show that PGE2 activates background 2 pore K+ channel TRESK, leading to hyperpolarization. We hypothesize that HF feeding, acting through the PG pathway, triggers cyclic AMP synthesis in the NG, which in turn, activates a novel Epac-Rap-PLCe pathway to stimulate the calcium-sensitive 2 pore K+ channel TRESK. This results in hyperpolarization and decreased vagal responsiveness to satiety signals. To test this hypothesis we have 4 specific aims. Aim 1 is to demonstrate that HFD modifies basic electrophysiological properties of NG neurons. Patch clamp recordings will be performed to characterize the excitability of NG ganglia neurons from control and rats fed with HFD. Physiological implications of these abnormalities will be evaluated by in vivo electrophysiological recording of NG in rats fed with HFD and study its responsiveness to satiety hormones such as CCK, leptin, bombesin and uroguanylin. Time course studies will be performed and changes will be correlated with metabolic gut permeability and ileal microbiota. Aim 2 examines whether hyperpolarization of NG neurons following HFD is mediated by activation of TRESK K+ channels. The presence of specific TRESK channels in NG will be identified using electrophysiological studies as well as Western blot and RT-PCR methods. The participation of the TRESK channel will be demonstrated by silencing the expression of TRESK channel in NG using siRNA technology. In vivo NG recording will be performed to examine the reversibility of electrophysiological abnormalities following silencing TRESK channel expression through electroporation of the NG with TRESK siRNA. Aim 3 investigates the signal transduction cascades that mediate the membrane modifications of NG neuron following HF feeding. We plan to perform simultaneous Ca2+ imaging studies and perforated patch electrophysiological recording of NG neurons to dissect which molecules are involved in mediating Ca2+ signaling resulting in cellular hyperpolarization. The importance of Epac, Rap and PLCe in the signal transduction cascade will be evaluated using siRNA technology. This will be complemented by Western blot and RT-PCR studies to demonstrate HFD results in upregulation of the Epac-Rap2B-PLCe and TRESK pathways in a time course study. Finally in Aim 4, we will perform feeding studies following silencing of key molecules in the cAMP-Epac-Rap-PLCe pathway. Understanding the abnormalities in the signal transduction cascade in the NG following HFD may provide important therapeutic targets for the prevention or correction of hyperphagia in patients consuming HF diets.